Summary of Work: The goals of this project are to understand the biochemistry and genetics of MMR in normal eukaryotic cells, and how mutations in MMR genes lead to environmentally associated human diseases. This year we investigated the mismatch repair function of a critical glutamate in the Phe-X-Glu motif of bacterial MutS and yeast Msh6. The major eukaryotic mismatch repair pathway requires Msh2-Msh6, which, like E. coli MutS, binds to and participates in repair of the two most common replication errors, single base-base and single base insertion-deletion mismatches. For both types of mismatches, the side chain of E. coli Glu38 in a conserved Phe-X-Glu motif interacts with a mismatched base both electrostatically and by forming a hydrogen bond with the N7 of purines or the N3 of pyrimidines. We show here that changing E. coli Glu38 to alanine results in nearly complete loss of repair of both single base-base and single base deletion mismatches. In contrast, a yeast strain with alanine replacing homologous Glu339 in Msh6 has nearly normal repair for insertion-deletion and most base-base mismatches, but is defective in repairing base-base mismatches characteristic of oxidative stress, e.g., 8-oxo-GYA mismatches. The results suggest that bacterial MutS and yeast Msh2-Msh6 differ in how they recognize and/or process replication errors involving undamaged bases, and that Glu339 in Msh6 has a specialized role in repairing mismatches containing oxidized bases. Additional work is in progress on the functions of several different MMR proteins, including yeast Msh6, Mlh1, Pms1 and mouse ExoI.[unreadable] [unreadable] Rare DNA synthesis errors are corrected by post-replication DNA mismatch repair (MMR). In addition to their functions in repairing replication errors, some eukaryotic MMR proteins also participate in other DNA transactions that are important for genome stability, toxicity and human health. These include critical environmental stress-response pathways such as repair of double-strand DNA breaks and DNA damage surveillance to signal apoptosis. MMR proteins also prevent recombination between DNA sequences with imperfect homology, they participate in meiotic recombination, and they modulate both somatic hypermutation of immunoglobulin genes and the stability or triplet repeat sequences whose instability is associated with certain hereditary degenerative diseases. Loss of MMR increases mutation rates and decreases apoptosis in response to certain forms of DNA damage, ultimately leading to cancer. Mutations in certain MMR genes result in infertility. The goals of this project are to understand the biochemistry and genetics of MMR protein function in normal eukaryotic cells, and how mutations in MMR genes lead to environmentally associated diseases.